In Vitro Photoprotective, Anti-Inflammatory, Moisturizing, and Antimelanogenic Effects of a Methanolic Extract of Chrysophyllum lucentifolium Cronquist

UVB exposure causes DNA mutation and ROS generation, which lead to skin photoaging, skin wrinkling, skin sagging, and uneven skin pigmentation. ROS activate the NF-κB and MAPK signaling pathways leading to production of inflammatory molecules such as COX-2, collagen-degrading proteins such as matrix metalloproteinases (MMPs), and moisture-deficiency-related proteins such as hyaluronidases (HYALs). UVB exposure also induces irregular skin pigmentation though melanin overproduction, related to CREB transcription factor activity and transcription of melanogenesis genes. Here, we demonstrate that Chrysophyllum lucentifolium methanol extract (Cl-ME) has antioxidant activity; it dose-dependently decreased the expression of COX-2, MMP-1, MMP-9, HYAL-1, and HYAL-4 by downregulating the NF-κB (IKKα/β, IκBα) and MAPK (ERK, JNK, and p38) pathways and increased the expression of Col1a1, which encodes a protein important for maintaining skin elasticity. Cl-ME also showed promising antimelanogenic activity by decreasing the expression of CREB, a transcription factor, which in turn inhibited the expression of genes encoding tyrosinase, MITF, TYRP1, and TYRP2. In summary, a methanol extract of C. lucentifolium exhibited antiphotoaging and antimelanogenic activity and could be useful in the cosmeceutical industry.


Introduction
Skin degradation is caused by both intrinsic and extrinsic aging. Intrinsic skin aging is mainly due to genetic changes, while extrinsic aging occurs through continuous exposure to environmental agents [1]. Solar exposure is the most important extrinsic aging factor, and photoaging is caused by UV radiation. UV radiation can be divided into three types by wavelength: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm) [2,3]. UVB is generally considered the most skin-damaging of the three because it causes sunburn, inflammation, and erythema and deeply penetrates the skin, resulting in DNA mutation and generation of radical oxygen species (ROS) [4]. ROS are involved in initiation and conduction of a cascade of signaling events that lead to skin photoaging, skin aging, and wrinkle formation [5].
The cascade of events that occurs in skin after exposure to UVB radiation and consequent ROS generation includes activation of NF-κB, a transcription factor that interacts with IκB-kinase (IKK), triggering phosphorylation of IκB, translocation of NF-κB into the nucleus, and subsequent expression of genes involved in the UVB-induced inflammatory response [6]. Recent studies have shown that activation of the mitogen-activated protein kinase (MAPK) pathway, which includes the activity of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, is strongly correlated with the process 2. Results 2.1. HPLC Analysis of Cl-ME and Its Cytotoxicity toward HaCaT, HDF, HEK293T, and B16F10 Cells The cytotoxicity of a methanol extract of C. lucentifolium (Cl-ME) was tested in HaCaT, HDF, HEK293T, and B16F10 cells using the MTT assay. Cl-ME exhibited weak or slight cytotoxic activity at 150 and 200 µg/mL toward HaCaT (Figure 1a Figure 1d) cells less than 15 to 20%. To identify the active biochemical ingredients, we tested Cl-ME for luteolin, kaempferol, and quercetin, the well-known antioxidant compounds. However, according to HPLC analysis, none of these were present in Cl-ME (Figure 1e).

Radical Scavenging Activity and Protective Effect of Cl-ME against Cell Damage from UVB and H 2 O 2 in HaCaT Cells
As previously explained, UVB exposure causes DNA mutation and ROS generation, which lead to photoaging, wrinkling, and sagging of skin [22,23]. Morphological analysis was employed to explore the protective effects of Cl-ME against UVB and H 2 O 2. As shown in Figure 2a,b, UVB irradiation induced morphological changes and approximately 60% cell death. Cl-ME treatment attenuated the UVB-induced cell damage in a dose-dependent manner and raised cell viability from 40% to over 80%. A similar pattern was observed upon H 2 O 2 treatment (Figure 2c,d). Therefore, 150 µg/mL of Cl-ME was selected for subsequent experiments. Next, HaCaT cells were stained with H 2 -DCFDA and DAPI. UVB irradiation enhanced ROS generation and DNA damage, while Cl-ME alleviated ROS-and UVB-mediated cell damage (Figure 2e). We used the ABTS assay to explore the free radical scavenging ability of Cl-ME, with ascorbic acid as the positive control. Cl-ME exhibited dose-dependent radical scavenging activity (Figure 2f).  HaCaT cells after a 24 h treatment with Cl-ME. (e) Protective activity of Cl-ME against ROS and cell damage as measured through DCFDA (10 µM) and DAPI (1 µL/mL) staining in HaCaT cells after 24 h of treatment. (f) Radical scavenging activity of Cl-ME was measured using the ABTS assay with ascorbic acid as the positive control. ## p < 0.01 compared to the normal group and ** p < 0.01 compared to the control group.

The Effect of Cl-ME on Moisture and Collagen
To examine the effect of Cl-ME on inflammatory markers, the expression of COX-2 was measured in the presence or absence of UVB (H 2 O 2 ) or Cl-ME. Cl-ME dose-dependently reduced the expression of the COX-2 gene in both UVB (30 mJ/cm 2 ) (Figure 3a)-and H 2 O 2treated (100 µM) HaCaT cells (Figure 3b). Matrix metalloproteinases and hyaluronidases degrade extracellular matrix proteins, leading to alterations such as skin wrinkling and aging [24]. UVB and H 2 O 2 promoted MMP and HYAL activity, which accelerate the formation of skin wrinkles (Figure 3c,d). Meanwhile, Cl-ME dose-dependently reduced the production of MMP-1, MMP-9, HYAL-1, and HYAL-4 in UVB (30 mJ/cm 2 ) (Figure 3c)and H 2 O 2 -treated (100 µM) HaCaT cells (Figure 3d). Another molecule important in maintaining skin elasticity is collagen, which is the main component of the ECM and whose degradation diminishes skin flexibility, tensile strength, and ability to hold water [12]. In this case, Cl-ME not only attenuated the UVB (30 mJ/cm 2 )-and H 2 O 2 -induced decrease in Col1a1 expression in HDF cells (Figure 3e,f) but also showed an intrinsic ability to increase collagen levels in HDF cells (Figure 3g). Moreover, luciferase reporter assay showed that Cl-ME and retinol increased luciferase activity at the transcriptional level ( Figure 3h).

Cl-ME Downregulated the Inflammation-Associated NF-kB and AP-1 Pathways
UVB is responsible for activating numerous signaling cascades, such as the NF-κB and AP-1 pathways [25]. Initiation of the NF-κB and AP-1 is known to regulate the activity of MMPs and COX-2. We therefore investigated the effect of Cl-ME on NF-κB and AP-1 pathways. Cl-ME inhibited PMA-induced luciferase activity mediated by NF-κB and AP-1 (Figure 4a,b). Furthermore, to investigate the regulatory mechanism of Cl-ME, the phosphorylation level of proteins upstream of NF-κB (IKKα/β, IκBα) and AP-1(ERK, p38, JNK) was measured. Phosphor forms of IKKα/β, IκBα, ERK, p38, and JNK were dramatically induced by UVB irradiation and significantly suppressed by Cl-ME treatment in a dosedependent manner (Figure 4d,e).

Antimelanogenic Effect of Cl-ME in B16F10 Cells through Downregulation of the CREB Pathway
Human skin pigmentation is controlled by a process called melanogenesis, which plays an important role in preventing skin injury from UV radiation [13]. To examine the effect of Cl-ME on melanogenesis, B16F10 cells were induced by α-MSH and then treated with Cl-ME or arbutin. Arbutin was used as a positive control because it inhibits melanin biosynthesis by decreasing tyrosinase activity. The level of melanin secreted was increased four-fold by α-MSH treatment but was reduced to about one-quarter by Cl-ME (150 µg/mL). The inhibitory effect of Cl-ME on α-MSH-stimulated melanin production was less dramatic. Cl-ME (150 µg/mL) treatment decreased the melanin content by 20%. To investigate whether Cl-ME-mediated antimelanogenesis is related to enzyme activity, the activity of tyrosinase, a vital kinase in melanin synthesis, was determined [26]. A mushroom tyrosinase assay illustrated that Cl-ME (150 µg/mL) treatment decreased tyrosinase activity by 30%; in comparison, kojic acid, a known tyrosinase inhibitor, suppressed tyrosinase activity by 43% [27]. Many signaling molecules modulate the melanogenic process, including tyrosinase enzyme, TYRPs, and their transcription factors, such as MITF and CREB [28]. Luciferase reporter assay showed that Cl-ME (150 µg/mL) decreased the transcriptional activity of CREB in a dose-dependent matter (Figure 5d). Furthermore, we observed a reduction in the expression of melanin production-related proteins such as MITF, TRP-1, and TRP-2 upon Cl-ME treatment (Figure 5e

Discussion
Plants in the genus Chrysophyllum are edible and have multiple health benefits. Chrysophyllum abidum fruit exerts anti-inflammatory activity by inhibiting NF-κB activation [29]. Chrysophyllum caimito stem bark extract was shown to have an antitumorigenic effect in hepatocellular carcinoma by promoting apoptosis of Hep G2 cells [30]. This study aimed to investigate if Chrysophyllum lucentifolium, a shrub in the genus Chrysophyllum, had a protective effect against UVB-or H 2 O 2 -induced cell damage and to explore the underlying mechanisms.
Our skin is exposed to ultraviolet light during outdoor activities, which can cause damage. Continuous exposure to UV can induce inflammation, resulting in production of a range of inflammatory cytokines and keratinocyte death. Excessive UV exposure triggers the accumulation of ROS, leading to oxidative stress, DNA mutation, skin aging, and development of skin cancers. A vital stage in choosing a compound suitable for use as a cosmeceutical compound is determining its cytotoxicity; minimal toxicity is critical for pharmaceutical and cosmetic formulations [31]. Cl-ME was toxic at concentrations of 200 µg/mL toward HaCaT (Figure 1a) and B16F10 cells (Figure 1d) but showed no significant cytotoxicity at the same concentration toward HDF (Figure 1b) and HEK293T cells (Figure 1c). Therefore, a concentration of 150 µg/mL of Cl-ME was chosen for all subsequent experiments.
Antioxidant compounds inhibit ROS generation and impact skin aging positively. An important step in the search for promising antiaging compounds is to determine the presence or absence of antioxidant activity [32]. We tested Cl-ME for the presence of luteolin, kaempferol, and quercetin, which are well-known antioxidant compounds. HPLC analysis failed to detect the presence of these compounds in Cl-ME (Figure 1e). Therefore, its antioxidant, antimelanogenic, and antiaging activities are apparently due to other intrinsic compounds. Coumaric acid [19], spinasterol, 6-hydroxyflavanone, 4dihydroxybenzoic acid, taraxerol, taraxerone, and lupeol acetate [32] have been found in plants in the Sapotaceae family and have antioxidant and antiaging effects. A more extensive analysis of compounds present in Cl-ME is required to identify components responsible for the antioxidant, antimelanogenic, and antiaging activity of this extract.
As previously described, UVB exposure causes DNA mutation and ROS generation, which lead to photoaging, wrinkling, and sagging of skin [22,33]. Thus, a photoprotective compound should have inherent radical scavenging activity so that it can prevent cell damage and death caused by both UVB radiation itself and the UVB-induced generation of ROS. Consistent with previous studies, UVB exposure and H 2 O 2 treatment resulted in the death of a large number of keratinocytes as well as morphological changes. Cl-ME had a photoprotective effect; it decreased the percentage of dead cells (Figure 1b,d). Additionally, Cl-ME had significant, dose-dependent ROS and cell-damage inhibitory activity in UVB-treated HaCaT cells (Figure 2e). Moreover, Cl-ME had important, dosedependent ABTS radical scavenging activity (Figure 2f). Thus, it helped to prevent both ROS-and UVB-mediated damage to cells.
UV radiation increases the expression of COX-2, MMP, and HYAL in the skin, all of which play an important role in skin photoaging. UVB-mediated overexpression of COX-2, for example, causes inflammation, itching, and oxidative DNA damage, which eventually lead to photoaging symptoms [22,34]. An increase in the expression of MMPs causes skin photoaging through degradation of ECM proteins such as collagen, elastin, and fibronectin [24]. UVB-mediated overproduction of hyaluronidases plays a part in skin aging as well because degradation of hyaluronic acid, a water-retaining molecule important in maintaining skin hydration, leads to skin wrinkling, sagging, and generalized aging [35]. Cl-ME reduced the expression of COX-2 in both UVB-and H 2 O 2 -treated (100 µM) HaCaT cells (Figure 3a,b). The antiaging effect of Cl-ME was not only observed through its ability to decrease the levels of MMPs and HYALs upon UVB and H 2 O 2 stimulation, but also its ability to promote collagen synthesis (Figure 3e-g). All these results suggest that Cl-ME is potentially useful in the cosmeceutical industry as an anti-inflammatory and antiaging compound.
AP-1, a transcription factor, is activated by upstream molecules (ERK, JNK, p38), while NF-κB is activated by IKKα/β and IκBα. Upon UVB irradiation, AP-1 and NF-κB are activated and induce the production of a series of cascade mediators. For instance, MMPs and COX-2 degrade collagen and other ECM components in the skin [9] and cause inflammatory responses that contribute to skin aging [36]. Because activation of those pathways is closely related to skin aging, Cl-ME is a promising antiaging compound because it dose-dependently attenuated the expression of signaling molecules in the AP-1 and NF-κB pathways based on luciferase assays and immunoblotting (Figure 4). Our results demonstrated that the anti-inflammatory and antiaging effects of Cl-ME are mediated by its targeting of the AP-1 and NF-κB pathways.
Overactivated melanogenesis can damage DNA and other molecules, alter immunological responses, and cause uneven skin pigmentation and aging [28]. Melanin production is closely related to activation of CREB transcription by α-MSH, which activates MITF and results in the expression of melanin-producing proteins such as TRP-1, TRP-2, and tyrosinase [28]. Current cosmetic trends have increased the demand for compounds that can attenuate melanogenesis. Cl-ME is a candidate for whitening cosmeceutical preparations because it decreased both melanin secretion and melanin content in B16F10 cells, diminished the activity of tyrosinase, and decreased the transcriptional activation of CREB. Additionally, expression of MITF, TRP-1, and TRP-2 genes and p-CREB, MITF, and tyrosinase proteins was blocked by Cl-ME ( Figure 5). All these results confirm that Cl-ME has significant antimelanogenic activity and thus high potential value in the cosmeceutical industry.

High-Performance Liquid Chromatography (HPLC)
To determine the compound present in Cl-ME, an Agilent 1260 HPLC system was employed. Luteolin, quercetin, and kaempferol were used as standard compounds in highperformance liquid chromatography. Separation was performed using a C18 reverse-phase chromatography column (250 mm × 4.6 mm, 5 µm). Mobile phase A was 0.1% acetic acid, and mobile phase B was 100% acetonitrile. Constant flow rate was 1.0 mL/min with gradient elution. Injection volume was 10 µL. UV absorbance was monitored at 350 nm, as described previously [46].

Cell Culture
HaCaT cells and B16F10, HDF, and HEK293T cells were cultured in media with 10% or 5% FBS, respectively. All cells were maintained in DMEM with 1% penicillin-streptomycin at 37 • C in a 5% humidified incubator.

Cell Viability Assessment
HaCaT, HDF, B16F10, and HEK293T cells were seeded in 96-well plates. After a 24 h incubation, cells were treated with Cl-ME (0-150 µg/mL) for 24 h or 48 h. The viability of each cell line was assessed using the MTT assay.

ABTS Radical Scavenging Activity
To determine the radical scavenging activity of Cl-ME, the ABTS assay was performed as reported previously [47]. A mixture of 7.4 mM ABTS and 2.4 mM potassium persulfate was prepared and incubated at room temperature overnight. Indicated doses of Cl-ME (0-150 µg/mL) or ascorbic acid were reacted with ABTS solution. The mixture was transferred to a 96-well plate and incubated at 37 • C for 30 min. The absorbance at 730 nm was read, and the ABTS scavenging effect was calculated as follows: where A 0 is the absorbance of ABTS, and A 1 is the absorbance [48].

Post UVB and H 2 O 2 Treatment Morphological Evaluation
For UVB and H 2 O 2 treatment, HaCaT cells were seeded in a 6-well plate at a density of 2 × 10 5 cells/mL, incubated with Cl-ME for 30 min, washed with PBS, and exposed to either H 2 O 2 (100 µM) or UVB radiation from a UVB lamp (30 mJ/cm 2 ). After H 2 O 2 or UVB treatment, cells were treated with Cl-ME (0-150 µg/mL) for 24 h. As previously reported [49], morphological changes and cell quantity were assessed using a microscope and ImageJ software. Cell survival was determined as follows: Cell survival (% of control): A 1 /A 0 × 100 where A 1 refers to treated cells and A 0 to normal untreated cells.

Semi-Quantitative RT-PCR Analysis
Cells were treated with UVB and H 2 O 2 as described above. Cl-ME (0-150 µg/mL) was then added to cells for 24 h. To measure the expression of melanin production-related genes, B16F10 cells were seeded at a density of 1 × 10 5 cells/mL in 12-well plates. After 24 h, cells were treated with α-MSH (100 nM), Cl-ME (0-150 µg/mL), or arbutin (1 mM), followed by a 24 h incubation. Total mRNA was isolated using TRIzol reagent according to the manufacturer's instructions [48]. cDNA was synthesized using a cDNA synthesis kit, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using the primers listed in Tables 1 and 2.

ROS and DAPI Staining
HaCaT cells were treated as explained above. After UVB treatment, cells were washed with PBS, stained with 10 µM DCFDA, and incubated for 30 min in the dark. Then, cells were fixed in formaldehyde solution (100 µL/mL) and prepared for DAPI staining. ROSstained and fixed cells were washed twice with PBS, stained with DAPI (1 µL/mL), and maintained in the dark for 20 min, as described previously [19].

Melanin Secretion and Content Assay
B16F10 cells were treated with α-MSH (100 nM), Cl-ME (0-150 µg/mL), or arbutin (1 mM) and incubated for an additional 48 h. Determination of melanin secretion and content was conducted according to a previous report [50]. Pictures were taken to assess melanin production as a color change, and melanin secretion and melanin content were measured at an absorbance of 475 nm and 405 nm, respectively. Both melanin secretion and content were expressed as percentage, as follows: Melanin secretion/melanin content % = A 1 /A 0 × 100 where A 1 is the mean absorbance of the sample, and A 0 is the mean absorbance of the control group.

Tyrosinase Assay
For the tyrosinase assay, 50 µL of 6 mM L-DOPA (previously dissolved in 50 mM potassium phosphate buffer, pH 6.8) was combined with 50 µL of Cl-ME or kojic acid and reacted for 15 min at room temperature. Afterward, 50 µL of mushroom tyrosinase (100 units/mL) was added, and the absorbance of the mixture was measured at 475 nm. Tyrosinase activity was calculated as follows: Tyrosinase activity % = A 1 /A 0 × 100 where A 1 is the mean absorbance of the sample, and A 0 is the mean absorbance of the control group.

Plasmid Transfection and Luciferase Reporter Gene Assay
HEK293T and B16F10 cells were seeded in 24-well plates. Transfection with luciferaseexpressing genes (NF-κB, AP-1, and CREB) was carried out using the PEI method for 24 h. Cells were incubated with different concentrations of Cl-ME for 24 h. Absorbance was measured using a luciferase assay system, and a β-galactosidase construct (0.1 µg/well) was used as the control.

Western Blot Analysis
HaCaT and B16F10 cells were treated with Cl-ME (0-150 µg/mL) for 30 min, washed with PBS, and subjected to UVB treatment (30 mJ/cm 2 ). After UVB treatment, cells were washed with PBS and treated for 24 h with Cl-ME (0-150 µg/mL). For B16F10 cells, induction was carried out as explained previously. Cell lysates were prepared and then subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Using specific antibodies, total and phosphorylated forms of target proteins were detected and visualized using chemiluminescent reagents [51].

Statistical Analysis
All data are presented as mean ± standard deviation (SD) of at least three independent experiments. A Mann-Whitney test was used to compare statistical differences between the experimental and control groups, and a p value < 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01). All statistical analyses were conducted using SPSS (SPSS Inc., Chicago, IL, USA).

Conclusions
In summary, we demonstrated that Cl-ME has significant antioxidant activity, antiaging effects, and anti-inflammatory effects due to dose-dependent inhibition of the expression of COX-2, MMP-1, MMP-9, HYAL-1, and HYAL-4 through downregulation of NF-κB (IKKα/β, IκBα) and AP-1 signaling pathway proteins (ERK, JNK, p38), as summarized in Figure 6. In addition, Cl-ME increased the expression of Col1a1, which is important for maintaining skin elasticity. Furthermore, Cl-ME showed promising antimelanogenic activity by decreasing the expression of CREB, which resulted in a reduction in the expression of melanogenesis-related genes such as tyrosinase, MITF, TRP-1, and TRP-2. We conclude that Cl-ME is a potentially valuable cosmeceutical ingredient due to its antiaging and antimelanogenic activities. Figure 6. Schematic pathway illustrating the moisturizing, antiaging, anti-inflammatory, and antimelanogenic effects of Cl-ME in vitro. Upon UVB, H 2 O 2 , or α-MSH stimulation conditions, AP-1, NF-κB, and CREB pathways are initiated. By these pathways, expression of MMPs, COX-2, HYALs, and tyrosinase is increased, leading to skin inflammation, collagen degradation, and moisturizing deficiency, as well as pigmentation. Cl-ME plays its role in moisturizing, antiaging, anti-inflammatory, and antimelanogenic effects by targeting AP-1, NF-κB, and CREB pathways.